In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses.

A rabbit animal model of the human disease familial hypercholesterolemia (FH), which is the result of low density lipoprotein (LDL) receptor deficiency, was used to develop an in vivo approach to gene therapy based on recombinant adenoviruses. Recombinant, replication-defective adenoviruses expressing the lacZ gene under the control of different promoters were infused into the portal circulation of New Zealand White (NZW) rabbits. Expression of lacZ could be obtained in virtually all hepatocytes within 3 days post-infusion, but was undetectable by 3 weeks. This was not associated with liver pathology. An LDL receptor expressing adenovirus was constructed using the most active promoter and was infused into the portal vein of rabbits deficient in LDL receptor. Analysis of liver tissues harvested 3 days after virus infusion demonstrated human LDL receptor protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold. Transgene expression was stable for 7-10 days and diminished to undetectable levels within 3 weeks. Infusion of LDL receptor expressing virus led to substantial reductions in serum cholesterol that returned to base line within 3 weeks; this acute reduction in serum cholesterol was associated with accumulations of lipid in hepatocytes. The development of neutralizing antibodies to the recombinant adenovirus markedly diminished the effectiveness of a second dose. These studies illustrate the advantages of recombinant adenoviruses for the treatment of liver metabolic diseases and define issues, such as viral genome instability and blocking immune response, that need to be overcome before the promise of this technology can be fully realized.

Electron microscopic definition of intestinal endocrine cells: immunogold localization and review.

Intestinal mucosal biopsy specimens processed during the past 25 years were used to example the ultrastructural characteristics of intestinal endocrine cells. The cells were defined on the basis of morphologic criteria and, when feasible, with specific antisera and immunogold staining. The hypothesis was that each endocrine cell, once well defined, should be identifiable on the basis of standard morphologic criteria not requiring specific immunostaining. This was not the case, D, G, EC1, EC2, ECn, D1, and intestinal gastrin cells have characteristic secretory granules and, when sufficient granules are present, can be identified consistently on the basis of morphologic criteria. Absolute identification of D, G, IG, and TG cells requires staining with specific antisera, a condition easily obtainable only for D, G, and IG cells. D1, EC1, EC2, and ECn cells must be identified morphologically until secretory products specific for each of these cells are identified. I, L, N, and K cells are remarkably similar in appearance and must be distinguished by specific staining. Mo, S, and P cells were not identified by either morphologic appearance or immunostaining. It is suggested that a cell similar to the D1 cell but with exceptionally small granules may be the P cell. Absolute identification of intestinal enteroendocrine cells by electron microscopy requires specific staining. The characteristic appearance of the secretory granules of many of these cells (D, G, EC1, EC2, ECn, D1, and IG) permits morphologic identification when numerous secretory granules are present.

Studies of the rectal mucosa in coeliac sprue: the intraepithelial lymphocyte.

The dynamics of the rectal surface epithelial lymphocyte and leucocyte response to wheat, gluten, and gliadin enema challenges in control individuals and in patients with coeliac sprue in remission is shown. There is a clear increase in intraepithelial lymphocytes and polymorphonuclear (PMN) leucocytes in response to these enemas in coeliac sprue, but not in controls. The peak response was at eight hours and cleared within 24 hours. There was no change in the crypt epithelium. These data add further support to the role of wheat, gluten, and gliadin in the pathogenesis of coeliac sprue, at least in the rectal mucosa.

Immunoelectron microscopic localization of HLA-DR antigen in control small intestine and colon and in inflammatory bowel disease.

We have elucidated the distribution of I2 (HLA-DR) antigen in control and inflammatory bowel disease specimens, using immunoelectron microscopic methods. Control small intestinal epithelium and inflammatory bowel disease epithelium expressed 12 antigen, while control colonic epithelium did not. I2 expression by enterocytes was more frequent on the lateral and basal surface than on the microvillus surface. Two of three M cells in control ileum expressed I2 antigen. I2-positive intraepithelial lymphocytes were rarely detected in both control and disease specimens. I2-positive lamina propria lymphocytes were significantly increased in inflammatory bowel disease, while I2-positive lamina propria lymphocytes were virtually absent in control specimens. I2-positive mononuclear cells in the intestinal lamina propria were largely macrophages and monocytes in both control and inflammatory bowel disease specimens. I2-positive mononuclear cells resembling dendritic cells were not detected in control or disease specimens. Furthermore, there were no significant morphological differences in I2-positive or -negative macrophages and monocytes in control and disease specimens. The expression of I2 antigen on Schwann cells was detected more frequently in disease specimens than in control specimens. Capillary endothelia of both control and disease specimens expressed I2 antigen. We demonstrate that I2 expression is present on surface membranes of both immune and nonimmune cells of the intestine and colon and show that this expression is more prominent in inflammatory bowel disease than in control intestine and colon. Further studies are required to determine whether this finding is meaningful in terms of antigen presentation and whether this apparent "immune activation" is involved in the pathogenesis of inflammatory bowel disease.

Classification and treatment of chronic nonhealing wounds. Successful treatment with autologous platelet-derived wound healing factors (PDWHF).

Previous animal data showed that platelets contain growth factors that stimulate capillary endothelial migration (angiogenesis), fibroblast proliferation and migration, and collagen synthesis. This study utilized autologous platelet-derived wound healing factors (PDWHF) to treat 49 patients with chronic nonhealing cutaneous ulcers. Patients were classified on the basis of 20 clinical and wound status parameters to generate a wound severity index. Forty-nine patients--58% diabetic (20% with renal transplants); 16% with trauma, vasculitis, etc.; 14% with decubitus ulcers; and 6% each with venous stasis or arterial insufficiency--with a total of 95 wounds had received conventional wound care for an average of 198 weeks (range: 1-1820 weeks). After informed consent was obtained, patients received autologous PDWHF. Mean 100% healing time for all patients was 10.6 weeks. There was no abnormal tissue formation, keloid, or hypertrophic scarring. A multivariant analysis showed a direct correlation to 100% healing with initial wound size and the initiation of PDWHF therapy. This is the first clinical demonstration that locally acting growth factors promote healing of chronic cutaneous ulcers.

Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease.

Using monoclonal antibodies to T and B lymphocytes, to natural killer cells, and to HLA-DR antigen, we characterized the lymphocyte population within the epithelial and lamina propria regions in control intestine and colon, and in grossly involved and in grossly uninvolved intestine and colon of patients with active inflammatory bowel disease. There were significantly more intraepithelial T cells in control ileum than in control colon. In comparison to control, there was a heterogeneity of alterations in intraepithelial and lamina propria T lymphocyte subsets (T11+, T8+, T4+) in inflammatory bowel disease. B lymphocytes were not detected within the lamina propria, except when found in and adjacent to lymphoid aggregates. Leu 7+ cells were uncommon in the lamina propria of control ileum and colon and in diseased tissues. The majority of intraepithelial lymphocytes did not express HLA-DR. Epithelial cells of control colon did not express HLA-DR while epithelial cells of control ileal tissues and of diseased colonic and ileal specimens expressed HLA-DR antigen. Only small numbers of lamina propria T cells expressed HLA-DR in both control and disease tissues. There was intense expression of HLA-DR by monocytes and modest expression of HLA-DR by capillary and lymphatic endothelial cells. The induction of HLA-DR expression by diseased colonic epithelium and the observation that lymphatic endothelium expresses HLA-DR are new observations, and we established that Leu 7+ cells are present in very small numbers in both normal and diseased intestine and colon.

In vitro characterization of human intestinal intraepithelial lymphocytes.

Enriched populations of human intestinal and colonic intraepithelial lymphocytes were isolated separate from lamina propria lymphocytes. These populations, and peripheral blood mononuclear cells, were characterized for membrane features, lymphocyte subsets, and proliferative potential to the nonspecific mitogenic lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen. All three populations were predominantly T lymphocytes as measured by sheep red blood cell rosettes and monoclonal antibodies. Peripheral blood mononuclear cells had a T4:T8 ratio of 2.15 while intraepithelial lymphocytes were enriched for T8+ cells with a T4:T8 ratio of 0.06-0.14. Peripheral blood mononuclear cells and lamina propria lymphocytes registered vigorous proliferative responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Intraepithelial lymphocytes, in contrast, failed to respond to the lectins. Control experiments with peripheral blood mononuclear cells indicated that the isolation reagents and procedures had no adverse effect on lectin-stimulated proliferation or on T-cell subset proportions. Viability of the intraepithelial lymphocytes after separation (88%) and after 3 days in culture with lectin (77%-85%) was confirmed by trypan blue dye exclusion, by light microscopy, by electron microscopy, and by ability to affect pokeweed mitogen-induced immunoglobulin synthesis when added to autologous and heterologous peripheral blood mononuclear cells. The failure of intraepithelial lymphocytes to respond to lectins was not due to failure of lectin binding, macrophage depletion, or absence of T-cell growth factor in culture. The possibility that the T-cell subset proportions (T4:T8 ratio) affect the proliferative response of isolated intraepithelial lymphocytes is discussed.

Intraepithelial leukocytes of the intestinal mucosa in normal man and in Whipple's disease: a light- and electron-microscopic study.

Intraepithelial lymphocytes (IEL) of the intestinal mucosa of normal man and of patients with Whipple's disease were studied by light microscopy of 1-micron-thick sections, and by electron microscopy of thin sections. IEL in normal human intestine tend to be elongated in outline, have few cytoplasmic organelles, have compact nuclei, and are unattached to epithelial cells. IEL in Whipple's disease are more likely to be activated in appearance, ie, to be larger and to contain more cytoplasmic organelles than IEL of normal intestine. The number of IEL/100 intestinal epithelial cells is similar in normal man and in patients with Whipple's disease. Other intraepithelial (IE) cells found in normal intestine include eosinophils and mast cells, and we note for the first time the presence of IE macrophages. There are no "globule leukocytes" in the intestine of normal man or of patients with Whipple's disease. Other IE cells found in the intestine in Whipple's disease include eosinophils, polymorphonuclear (PMN) leukocytes, and macrophages in untreated disease and intraepithelial macrophages in treated disease. These IE cells may be involved in the acute and chronic immune responses of the intestine.